PAL is a non-mammalian enzyme widely distributed in plants (Koukol, et al., J. Biol. Chem. 236:2692-2698 (1961); Hanson, et al., The Enzymes 7:75-166 (1972); Poppe, et al., (2003) ibid.), some fungi (Rao, et al., Can. J. Biochem. 4512:1863-1872 (1967); Abell, et al., Methods Enzymol. 142:242-253 (1987)) and bacteria (Bezanson, et al., Can. J. Microbiol. 16:147-151. (1970); Xiang, et al, J. Biol. Chem. 277:32505-32509 (2002); Hill, et al., Chem. Coomun. 1358-1359 (2003)) and can be recombinantly produced in Escherichia coli. 
A representative list of PALs includes: Q9ATN7 Agastache rugosa; O93967 Amanita muscaria (Fly agaric); P35510, P45724, P45725, Q9SS45, Q8RWP4 Arabidopsis thaliana (Mouse-ear cress); Q6ST23 Bambusa oldhamii (Giant timber bamboo); Q42609 Bromheadia finlaysoniana (Orchid); P45726 Camellia sinensis (Tea); Q9MAX1 Catharanthus roseus (Rosy periwinkle) (Madagascar periwinkle); Q9SMK9 Cicer arietinum (Chickpea); Q9XFX5, Q9XFX6 Citrus clementina×Citrus reticulate; Q42667 Citrus limon (Lemon); Q8H6V9, Q8H6W0 Coffea canephora (Robusta coffee); Q852S1 Daucus carota (Carrot); 023924) Digitalis lanata (Foxglove); O23865) Daucus carota (Carrot); P27991) Glycine max (Soybean); O04058) Helianthus annuus (Common sunflower); P14166, (Q42858) Ipomoea batatas (Sweet potato); Q8GZR8, Q8W2E4 Lactuca sativa (Garden lettuce); O49835, O49836 Lithospermum erythrorhizon; P35511, P26600 Lycopersicon esculentum (Tomato); P35512 Malus domestica (Apple) (Malus sylvestris); Q94C45, Q94F89 Manihot esculenta (Cassava) (Manioc); P27990 Medicago sativa (Alfalfa); P25872, P35513, P45733 Nicotiana tabacum (Common tobacco); Q6T1C9 Quercus suber (Cork oak); P14717, P53443, Q7M1Q5, Q84VE0, Q84VE0 Oryza sativa (Rice); P45727 Persea americana (Avocado); Q9AXI5 Pharbitis nil (Violet) (Japanese morning glory); P52777 Pinus taeda (Loblolly pine); Q01861, Q04593 Pisum sativum (Garden pea); P24481, P45728, P45729 Petroselinum crispum (Parsley) (Petroselinum hortense); Q84LI2 Phalaenopsis×Doritaenopsis hybrid cultivar; P07218, P19142, P19143 Phaseolus vulgaris (Kidney bean) (French bean); Q7XJC3, Q7XJC4 Pinus pinaster (Maritime pine); Q6UD65 Populus balsamifera subsp. trichocarpa×Populus deltoides; P45731, Q43052, O24266 Populus kitakamiensis (Aspen); Q8H6V5, Q8H6V6 Populus tremuloides (Quaking aspen); P45730 Populus trichocarpa (Western balsam poplar); O64963 Prunus avium (Cherry); Q94EN0 Rehmannia glutinosa; P11544 Rhodosporidium toruloides (Yeast) (Rhodotorula gracilis); P10248 Rhodotorula rubra (Yeast) (Rhodotorula mucilaginosa); Q9M568, Q9M567 Rubus idaeus (Raspberry); P31425, P31426 Solanum tuberosum (Potato); Q6SPE8 Stellaria longipes (Longstalk starwort); P45732 Stylosanthes humilis (Townsville stylo); P45734 Trifolium subterraneum (Subterranean clover); Q43210, Q43664 Triticum aestivum (Wheat); Q96V77 Ustilago maydis (Smut fungus); P45735 Vitis vinifera (Grape); and Q8VXG7 Zea mays (Maize).
Numerous studies have focused on the use of the enzyme phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) for enzyme substitution treatment of phenylketonuria (PKU) (Hoskins, et al., (1980) ibid.; Gilbert, et al., Biochem. J., 199(3):715-723 (1981); Hoskins, J. A., et al., (1982) ibid.; Sarkissian, et al., (1999) ibid.; Liu, et al., Artif. Cells Blood Substit. Immobil. Biotechnol., 30(4):243-257 (2002); Wieder, K. J., J Biol Chem., 254(24):12579-12587 (1979); Gamez, Development of Pegylated forms of Recombinant Rhodosporidium toruloides Phenylalanine Ammonia Lyase for the Treatment of Classical PKU. Mol. Ther. In press; Ambrus, et al., J. Pharmacol. Exp. Ther., 224(3):598-602 (1983); Ambrus, et al., Science, 201(4358):837-839 (1978); Kalghatgi, Res. Commun. Chem. Pathol. Pharmacol., 27(3):551-561 (1980); Ambrus, Res. Commun. Chem. Pathol. Pharmacol., 37(1):105-111 (1982); Gilbert, et al., Biochem. Biophys. Res. Commun., 131(2):557-563 (1985); Pedersen, Res. Commun. Chem. Pathol. Pharmacol., 20(3):559-569 (1978); Marconi, et al., Biochimie, 62(8-9):575-580 (1980); Larue, et al., Dev. Pharmacol. Ther., 9(2):73-81 (1986); Ambrus, C. M., et al., (1987) ibid.; Bourget, et al., Appl. Biochem. Biotechnol., 10:57-59 (1984); Bourget, et al., FEBS Lett., 180(1):5-8 (1985); Bourget, et al., Biochim. Biophys. Acta, 883(3):432-438 (1986); Chang, et al., Artif. Cells Blood Substit. Immobil. Biotechnol., 23(1):1-21 (1995); Chang, et al., Mol. Biotechnol., 17(3):249-260 (2001); U.S. Pat. No. 5,753,487).
PKU is an inborn error of amino acid metabolism that results from impaired activity of hepatic phenylalanine hydroxylase (PAH), the enzyme responsible for the metabolism of phenylalanine. Patients with PAH mutations that lead to PKU and hyperphenylalaninemia (HPA) display elevated levels of phenylalanine, impaired neurophysiological functioning and reduced cognitive development. For patients with severe PKU, there is the potential for irreversible mental retardation unless phenylalanine levels are maintained at low levels using dietary restrictions. PAL converts phenylalanine to ammonia and trans-cinnamic acid, a harmless metabolite, which is further metabolized and excreted in the urine as hippurate (Hoskins, Lancet 8165:392-394 (1980); Hoskins, et al., Biomed Mass Spectrom 11(6): 296-300 (1984)).
Current treatment for PKU involves the lifetime adherence to a diet that is low in the amino acid phenylalanine (Levy, Proc. Natl. Acad. Sci. U.S.A. 96(5):1811-1813 (1999)). This dietary therapy is difficult to maintain (Matalon, et al., Genet. Med., 6(1): 27-32 (2004); Woolf, et al., Arch. Dis. Child. 33(167):31-45 (1958); Kim, Mol. Ther. 10(2):220-224 (2004)) and does not always eliminate the damaging neurological effects that can be caused by elevated phenylalanine levels (Sarkissian, et al., Mol. Genet. Metab. 69:188-194 (2000)). Less than ideal dietary control during pregnancy can lead to birth defects (Levy, (1999) ibid.). In addition, it is very difficult for PKU/HPA patients to live a normal life while following the restrictive diet, and dietary therapy can be associated with deficiencies of several nutrients, some of which are detrimental for brain development (Levy, (1999) ibid.). Most low phenylalanine diet products have organoleptic properties sufficiently unsatisfactory that compliance with this treatment is compromised (Levy, (1999) ibid.). Therefore, development of a therapeutic treatment would replace or supplement the current dietary treatment and prevent the neurological damages inflicted on those individuals with PKU, particularly for those patients with the most severe forms of the disease.
In 1999, Scriver and colleagues reported their initial studies on the use of the enzyme PAL from Rhodosporidium toruloides (Sarkissian, et al., 1999 ibid.) for PKU enzyme substitution applications. Mouse PKU and HPA model studies demonstrated that PAL administration (either by i.p. injection or orally using either PAL in combination with aprotinin protease inhibitor or PAL recombinantly expressed and present inside E. coli cells) was associated with lower blood plasma phenylalanine levels. In addition, preliminary studies describing the use of PAL with PKU patients have shown reduction in phenylalanine levels using PAL administered in enteric-coated gelatin capsules (Hoskins, et al., (1980) ibid.) or using an extracorporeal enzyme factory (Ambrus, et al., Ann. Intern. Med., 106(4):531-537 (1987)). These studies suggest that if PAL is protected against proteolytic degradation, significant reductions of plasma Phe can be achieved upon oral administration.
The use of PAL for cancer treatment has also been suggested based on its ability to limit the nutrient supply of phenylalanine to cancer cells and thereby inhibit neoplastic growth (Fritz, et al., J Biol Chem. 251(15):726 (1976); Roberts, et al., Cancer Treat Rep., 60(3):261-263 (1976); Shen, et al., Cancer Res. 37(4):1051-1056 (1977); Shen, et al., Cancer Treat Rep. 63(6):1063-1068 (1979); Wieder, et al, J Biol Chem., 254(24):12579-12587 (1979)). However, intravenously injected pegylated PAL was cleared rapidly from circulating blood after the 13th injection. In addition, PAL-mediated reduction in phenylalanine prevented the proliferation of murine leukemia and metastatic melanoma (Abell, et al., Cancer Res. 33:2529-2532 (1973)), Roberts, et al., ((1976) ibid) Shen, et al., ((1977) ibid)).
Bacterial PAL from the marine bacterium Streptomyces maritimus may serve as an important source of the bacteriostatic agent enterocin. S. maritimus PAL, EncP, catalyzes the initial step in enterocin synthesis, which is the conversion of phenylalanine to trans-cinnamic acid (Xiang, et al., J. Biol. Chem. 277:32505-32509 (2002)).
PAL may be used for the industrial synthesis of L-phenylalanine methyl ester (for Aspartame production) (D'Cunha, et al., Enzyme and Microbial Technology 19(6):421-427 (1996); Hamilton, et al., Trends in Biotechnol. 3(3):64-68 (1985)) and other substituted L-phenylalanine derivatives that are used as pharmaceutical precursors (U.S. Patent App. 20020102712).
PAL may also have agricultural applications, wherein PAL participates in the initial enzymatic process leading to the phenylpropanoids that produce lignins, coumarins, and flavanoids in plants, fungi, and bacteria. Hence, modulation of PAL activity can influence a number of agricultural phenomena such as the browning of fruit. In addition, structure-based drug design of active site PAL inhibitors could lead to effective herbicides (Poppe, et al., ibid.).
Although PAL potentially has various industrial and therapeutic applications, the use of PAL may be limited by reduced specific activity and proteolytic instability. Similar to other therapeutic proteins, use of PAL as an enzyme therapy is accompanied by several disadvantages such as immunogenicity and proteolytic sensitivity. Further, a delicate balance is required between substrate affinity and enzyme activity to achieve and maintain control of plasma phenylalanine levels within a normal somewhat narrow range in disorders characterized by hyperphenylalanemia. As yet, a concerted effort toward improving these parameters has not been made due to a paucity of structural and biochemical knowledge regarding this protein.
Thus, there remains a need for PAL molecules with optimal kinetic characteristics including potent catalytic activity and greater biological half-life, greater biochemical stability and attenuated immunogenicity.